PROJECT SUMMARY Previous work under this P01 has demonstrated that a single treatment with the mitochondrial targeted peptide SS-31 improves skeletal muscle performance, mitochondrial function, and reduces redox stress. These surprising results demonstrate that mitochondrial dysfunction with age is a more dynamic process than previously thought and can be reversed by late-life treatment to improve healthspan. This new paradigm led directly to an ongoing clinical trial at the University of Washington led by Kevin Conley (PL-Core B) testing SS- 31 for improvement in skeletal muscle function in elderly humans. Preliminary data presented in this renewal also demonstrates that relatively short term treatment (4-8 weeks) with SS-31 can improve cardiac and skeletal muscle function and vision in aged rodents. Additional data from our lab, Core E, and others indicates that SS- 31 does not act as a traditional antioxidant by scavenging reactive oxygen species. Instead there is growing evidence that SS-31 interacts with mitochondrial cardiolipin to improve mitochondrial electron transport system (ETS) function and reduce mitochondrial oxidative stress. We propose that improved ETS function with short- term treatment reduces redox and energy stress which improves function and stress response of the aged muscle. With long-term treatment this improved stress signaling restores mitochondrial structure leading to further improvements in mitochondrial and skeletal muscle performance. Aim 1 uses normal aging and an ETS targeted toxin, doxorubicin, to test this hypothesis for the reversal of age-related muscle dysfunction. We assess the effect of 1 day, 1 week, and 8 week SS-31 treatment on the reversal of mitochondrial redox and energy stress, stress signaling regulating mitochondrial quality, and skeletal muscle performance. For short- term effects we will focus on targeted analyses of the thiol and phospho proteomes in key stress response and functional pathways. We have found that reducing mitochondrial oxidative stress reverses many of the age- related oxidative changes to the thiol proteome, including proteins involved in muscle contraction, EC coupling, protein quality control, and mitochondrial energetics. Aim 2 uses SOD1-/- mice to test whether protection of mitochondrial ETS function and reducing redox stress with SS-31 in this model of accelerated sarcopenia can prevent muscle atrophy when treatment is initiated at the onset of muscle dysfunction. Aim 3 is shared across projects and tests whether SS-31 treatment initiated at mid-life preserves healthspan in normal aging and in aging compounded by high fat diet. Many of the mechanisms tested in Project 2 are likely to be relevant to the aging heart (Project 1) and visual system (Project 3) as well. Therefore the parallel work in these three systems, in which we have already identified significant reversal of physiological decline with SS-31, provides the unique opportunity to identify aspects of mitochondrial function that contribute to the basic biology of aging across diverse physiological systems. The end result will be new insights into the mechanistic basis of this new paradigm for improving healthspan with potential for direct translation to elderly humans.